An electrochemical DNA biosensor for the detection of NOS gene sequences from genetically modified organisms (GMOs) is presented in this paper. Single-stranded DNA (ssDNA) was covalently attached through the carboxylate ester formed by the 3′-hydroxy end of the DNA with the carboxyl of a mercaptoacetic acid self-assembled monolayer-modified gold electrode using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) as linkers. The electrochemical behavior of methylene blue (MB) on the ssDNA and dsDNA modified gold electrode were carefully studied. Compared with ssDNA/Au electrode, an increase of redox peak current of MB on dsDNA/Au electrode was found, which could be further used for monitoring the recognition of DNA hybridization. Based on this result, the polymerase chain reaction (PCR) product of the common inserts NOS terminator from real GMOs samples was detected successfully.